Amyloid beta-peptide (Abeta) is central to the pathogenesis of Alzheimer's disease (AD), and the AD brain is under extensive oxidative stress. Previous research from our laboratory combined these two observations into a model for neurodegeneration in AD, a model based on Abeta-associated free radical oxidative stress. In neurons, Abeta-associated free radical oxidative stress and toxicity depend critically on methionine residue 35 of Abeta(1-42), and both are prevented or modulated by exogenous antioxidants. We have used the emerging techniques of proteomics, coupled with immunoblottmg methods, to identify specifically oxidatively modified proteins in AD brain. We now propose to extend these observations to sporadic and inherited AD and rodent models thereof to gain insight into the mechanisms of Abeta (1-42) in the oxidative stress and neurotoxic properties of this peptide. In Specific Aim # 1, we will use proteomics to test the hypothesis that there will be a common set of oxidized proteins in inherited and sporadic AD that differ from those of normal aging, and that these oxidized proteins in common are important in the pathogenesis of AD. Similar studies in mice models of inherited AD are hypothesized to lead to the identity of the same common proteins. In Specific Aim #2, we will test the hypothesis that mitochondrial dysfunction and Ca 2+ accumulation in brain from genetic mutations relevant to AD will be found in adult brain mitochondria from the model chosen in Specific Aim #1 compared to adult brain from wild-type mice. Those proteins identified by proteomics in the systems studied will be expressed in SY5Y cells and challenged with Abeta(1-42) to determine if there is increased vulnerability to oxidative stress, mitochondrial dysfunction, Ca 2+ accumulation, and cytotoxicity. In Specific Aim #3, we will use proteomics to test the hypothesis that Abeta(1-42)-induced oxidative stress in embryonic neuronal cultures obtained from the rodent model of familial AD chosen in Specific Aim # 1 leads to the same oxidatively modified proteins as exists in adult brain from this animal and in human familial AD brain. In Specific Aim #4, we will investigate whether oxidative stress induces mitochondrial alterations in neuronal cultures obtained from the rodent model of inherited AD chosen in Specific Aim #1, or whether mitochondrial alterations induced in these cultures leads to oxidative stress. In Specific Aim # 5, we will test the hypothesis that endogenous or exogenous antioxidants protect brain in-vivo against oxidative stress in the rodent model chosen in Specific Aim # 1. This is a comprehensive set of proposed studies, employing novel approaches in systems ranging from sporadic and inherited AD brain, to genetic animal models of AD, to neuronal cultures, designed to gain insight into Abeta-associated free radical oxidative stress and neurotoxicity and their modulation by endogenous and exogenous antioxidants. The increased understanding that will result from these studies will provide insight into potential therapeutic interventions in this important dementing disorder that affects millions of Americans.